An emerging view supports that steroid hormones, like many peptide growth factors, may possess both mitogenic and motogenic properties. This notion is backed by the recent finding that ecdysone regulates the embryonic border cell invasion in Drosophila. These results demonstrate that steroid hormones can stimulate invasive behavior independent of any discernable effects on cell proliferation. Tumor cell motility is a critical determinant in the progression of localized disease to metastasis. A novel p21-activated kinase 6 (PAK6) was recently identified to be an androgen receptor (AR) interacting protein. The expression of PAK6 is restricted to the hormone-sensitive tissues including prostate, testis and placenta. Initial characterization indicates that the interaction between PAK6 and AR is an androgen-dependent event. Most importantly, our recent results demonstrate an elevated expression of Pak6 associated with prostate cancer metastatic progression. Intriguingly, subcellular localization of PAK6 appears to be heterogeneous between normal and malignant epithelial;while PAK6 localized to the cell membrane in normal epithelia, intense cytoplasmic staining was observed in carcinoma cells. Although PAK6 does not appear to function as a cofactor in modulating AR transcriptional activity, results from our lab indicate its role in regulating hormone-dependent prostate cancer cell motility and survival. Ectopic expression of a PAK6 constitutive active mutant promotes cell motility and anchorage-independent soft agar cell growth of human prostate cancer LNCap cells. Conversely, expression of a PAK6 dominant negative kinase dead mutant inhibits cell motility and soft agar colony growth. Most intriguingly, these results further indicate that the interaction between PAK6 and AR is responsible, at least in part, for the activation of PAK6 in response to androgen stimulation in prostate cancer cells. Our model predicts that interaction between androgen receptor and PAK6 may play a pivotal role in controlling hormone-dependent prostate cancer metastasis. Hypothesis: Our working hypothesis is that androgen-dependent AR-mediated PAK6 activation promotes hormone-regulated metastatic phenotypes in prostate cancer. The goal of the current proposal is to define the physiological role of PAK6 in androgen receptor mediated prostate cancer progression. Specific Aims and Study Design: Aim 1: To determine the structural-functional requirement of the AR-PAK6 interaction in regulating androgen-stimulated PAK6 activation. This aim is designed to characterize the interaction between AR and PAK6 at the molecular level. The focus of this aim is to determine how PAK6 is activated by interacting with AR in response to androgen stimulation. The interacting domains of the two molecules will be mapped and critical residues contributing to the interaction will be identified. Conventional molecular biological techniques, mammalian two-hybrid assays and kinase assays will be employed. Aim 2: to identify and characterize molecular targets downstream of PAK6 pertinent to promote cell motility and invasion. A proteomic approach employing tandem affinity tagged PAK6 and tandem mass spectrometry will be used to identify PAK6 downstream candidate targets. Aim 3: To characterize the effect of PAK6 activation on LNCap cell tumorigenicity and metastasis using a nude mouse xenograft model. Relevance: The identification of PAK6 activation as an androgen-stimulated AR- mediated event suggests a potential target for intervention of hormone regulated prostate cancer metastasis. Fully dissecting the molecular interactions along the signal pathway will foster our long-term goal of development of new modality to control androgen-regulated prostate cancer metastasis. PUBLIC HEALTH RELEVANCE: Despite clinical evidence indicating that androgen promotes the metastatic progression of prostate cancer cells, the underlying mechanism remains unclear. The current proposal aims at studying a novel signal molecule PAK6 that functions as a dominant factor in controlling androgen-stimulated cell motility. The identification of PAK6 activation as an androgen-stimulated AR-mediated event suggests a potential target for intervention of hormone-regulated prostate cancer metastasis. Fully dissecting the molecular interactions along this signal pathway will fulfill the programmatic interest of understanding the basic biology between androgenic signals and prostate cancer metastatic progression.